Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China; Key Laboratory of Forensic Toxicology, Ministry of Public Security, Beijing, 100191, China.
Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China.
Anal Chim Acta. 2024 Nov 15;1329:343238. doi: 10.1016/j.aca.2024.343238. Epub 2024 Sep 11.
The apurinic/apyrimidinic (AP) site is an important intermediate in the DNA base excision repair (BER) pathway, having the potential of being a biomarker for DNA damage. AP sites could lead to the stalling of polymerases, the misincorporation of bases and DNA strand breaks, which might affect physiological function of cells. However, the abundance of AP sites in genomic DNA is very low (less than 2 AP sites/10 nts), which requires a sensitive and accurate method to meet its detection requirements. Here, we described an ultrasensitive quantification method based on a hydrazine-s-triazine reagent (i-PrN) labeling for AP sites combining with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The limit of detection reached an ultralow level (40 amol), realizing the most sensitive MS-based quantification for the AP site. To guarantee the accuracy of the quantitative results, the labeling reaction was carried out directly on DNA strands instead of labeling after DNA enzymatic digestion to reduce artifacts that might be produced during the enzymatic process of DNA strands. And selective detection was realized by MS to avoid introducing the false-positive signals from other aldehyde species, which could also react with i-PrN. Genomic DNA samples from different mammalian cell lines were successfully analyzed using this method. There were 0.4-0.8 AP sites per 10 nucleotides, and the values would increase 16.1 and 2.75 times when cells were treated with genotoxic substances methyl methanesulfonate and 5-fluorouracil, respectively. This method has good potential in the analysis of a small number of cell samples and clinical samples, is expected to be useful for evaluating the damage level of DNA bases, the genotoxicity of compounds and the drug resistance of cancer cells, and provides a new tool for cell function research and clinical precise treatment.
无碱基位点（AP）是 DNA 碱基切除修复（BER）途径中的一个重要中间产物，具有成为 DNA 损伤生物标志物的潜力。AP 位点可能导致聚合酶停滞、碱基错配和 DNA 链断裂，从而影响细胞的生理功能。然而，基因组 DNA 中 AP 位点的丰度非常低（少于 2 个 AP 位点/10 个核苷酸），这需要一种灵敏且准确的方法来满足其检测要求。在这里，我们描述了一种基于肼-均三嗪试剂（i-PrN）标记结合高效液相色谱-串联质谱（HPLC-MS/MS）的 AP 位点超灵敏定量方法。检测限达到超低位（40 飞摩尔），实现了基于 MS 的 AP 位点最灵敏定量。为了保证定量结果的准确性，标记反应直接在 DNA 链上进行，而不是在 DNA 酶切后进行标记，以减少 DNA 链酶切过程中可能产生的假象。通过 MS 实现选择性检测，避免了其他醛类物质与 i-PrN 反应产生的假阳性信号。使用该方法成功分析了来自不同哺乳动物细胞系的基因组 DNA 样本。每个 10 个核苷酸中有 0.4-0.8 个 AP 位点，当细胞分别用致突变物质甲磺酸甲酯和 5-氟尿嘧啶处理时，AP 位点的数量分别增加了 16.1 倍和 2.75 倍。该方法在少量细胞样本和临床样本的分析中具有良好的应用前景，有望用于评估 DNA 碱基的损伤水平、化合物的遗传毒性和癌细胞的耐药性，为细胞功能研究和临床精准治疗提供新的工具。
